The goal of the project consists in the development of a radically new technology for specific detection of DNA signature sites within genomic DNA in its native, double-stranded form. The new approach is based on the ability of peptide nucleic acid (PNA) to open up unique short sequences (20-30bp) within duplex DNA and make it possible to detect short DNA sequences within genomic DNA under non-denaturing conditions. On the site selectively opened by a pair of PNA openers, a circular oligonucleotide probe is assembled, which serves as a template for rolling circle amplification (RCA). The principal advantage of the approach to be developed consists in the enormous specificity, which entails two its major features. First, very short PNA oligomers are used, which result in zero tolerance to mismatches in the sites of DNA binding. Secondly, only sites opened by PNA openers are accessible to binding by circularazible oligonucleotides, which entails exceeding specificity of circular probe formation. The feasibility of this project is based on the data recently published by PI, in which similar approach has been successfully implemented for the development of a new method for bacterial detection. In the project these studies will be extended to detect chosen DNA target sites within double-stranded genomic DNA in eukaryotic cells including human cells. While adaptation of the approach to eukaryotic cells is expected to be much more challenging than in case of bacteria, the preliminary data on human nuclei are very promising. The approach to be developed will make it possible to target and specifically label individual genes on human chromosomes in situ, to detect various insertions and transpositions in chromosomes, including diseases-associated genomic instabilities, in a convenient FISH-like format. As a result of the project implementation, robust approaches for marking and detection of dsDNA under native conditions with potential wide applications in pathology will be developed, which will open new avenues in DNA diagnostics. Public Health Relevance Statement: The project's goal consists in development of a rapid, exceedingly specific and very sensitive approach for specific recognition, marking and localization of short DNA signature sites within genomic DNA in its native, double-stranded form. Its implementation carries great promise to equip biomedical and clinical researchers with new powerful tools for quick and reliable diagnosis. This diagnostic will be capable to target and specifically label individual genes on human chromosomes in situ, to detect various insertions and transpositions in chromosomes, including diseases-associated genomic instabilities, in a convenient FISH-like format.